Dan Kuppers 5/2011
(ref. Rothaeusler, K. and Baumgarth, N., Cytometry Part A 2006)

Materials

• BrdU 10 mg/mL, store frozen
• 10 million in vivo labeled thymocytes
• PBS w/o Ca or Mg
• PBS w/ Ca/Mg (PBS with 0.1 g/L CaCl2, 0.1 g/L MgCl2•6H2O)
• Wash Solution: PBS + 0.5% NP40
• Fixative: Fresh 1% paraformaldehyde + 0.05% NP40
• Quick 1% paraformaldehyde:

1. Mix 0.1g paraformaldehyde w/ 0.5mL H2O and one drop of 1N NaOH
2. Heat 2-3 min @ 80ºC untill disolved.
3. Add to 9.5 mL 1x PBS
4. Check that the pH is 7.4 (should not require adjusting)

• Cell surface antibody (e.g. PE conjugated Pharmingen antibodies diluted 1:100 - 1:50 in PBS)
• DNAse I (Invitrogen 212 U/µL)
• FITC-conjugated anti-BrdU antibody (Sigma, clone BU-33)
• Cell surface antibodies of interest
• 15 mL conical tubes
• Flow Cytometry tubes

Procedure

1. Label cells in vivo by injecting mice with 1mg of BrdU, i.p. 1 hr. prior to harvest. Include one no-BrdU mouse as a negative control.
2. Prepare a single cell suspension in PBS and count cells on a hemocytometer. Aliquot out 10 million thymocytes and spin at 1000-1500 rpm x5 min to pellet.
3. Discard supernatant and resuspend cells in 200 µL solution of conjugated cell surface antibodies of interest (1:50 - 1:100 in PBS) @ 4°C for 1hr. Note: Novel fluorophores should be be tested for loss of signal from fixation.
4. Wash 2x with PBS. If biotinylated antibodies were used then incubate cells in 200 µL of streptavidin-APC (1:100 in PBS).
5. Wash cells 2x with PBS. Loosen pellet and then incubate cells in 1mL Fixative o/n @ 4°C.

Next day

1. Wash cells 2x with PBS.
2. Resuspend cells in 250 µL PBS + Ca/Mg with 50 U/mL DNase I. Incubate @ 37°C for 30 min.
3. Wash 2X with Wash Solution and resuspend in 100 µL of a 1:5 PBS solution of FITC-conjugated anti-BrdU antibody. Incubate on ice for 45 min.
4. Wash with 1mL Wash Solution and resuspend the cells in 0.2 mL PBS (optionally with 10 µg/mL DAPI or PI) and transfer to flow cytometry tubes. If DAPI or PI is used incubate @ 4°C for 30 min. prior to flow cytometry.
5. Analyze the cells by Flow Cytometry. Use the no-BrdU control sample to set voltages. This is important since the anti-BrdU antibody gives a high background.

Tags: Fero-Flow, Fero-Lab-Protocols